Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Endothia parasitica protease hydrolyzes l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide at the peptide bond. l-Phenylalanyl-l-leticine amide, N-carbobenzoxy-l-leucyl-l-phenylalanine amide, N-carbobenzoxy-l-leucyl-l-pheml-alanine, N-carbobenzoxy-l-phenylalanyl-l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, vizl-leucyl-l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl-l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl-l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.     

Relation between Neutral Lipid Accumulation and the Growth Phase in the Yeast,Lipomyces starkeyi,a Fat Producing Yeast

In order to investigate the substrate binding feature of undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26 with respect to farnesyl diphosphate and a reaction intermediate, (Z,E,E)-geranylgeranyl diphosphate, we examined the reactivity of artificial substrate analogs, 3-desmethyl farnesyl diphosphate and 3-desmethyl Z-geranylgeranyl diphosphate, which lack the methyl group at the 3-position of farnesyl diphosphate and Z-geranylgeranyl diphosphate, respectively. Undecaprenyl diphosphate synthase did not accept either of the 3-desmethyl analogs as the allylic substrate, indicating that the methyl group at the 3-position of the allylic substrate is important in the undecaprenyl diphosphate synthase reaction. These analogs showed different inhibition patterns in the cis-prenyl chain elongation reaction with respect to the reactions of farnesyl diphosphate and Z-geranylgeranyl diphosphate as allylic substrate. These results suggest that the binding site for the natural substrate farnesyl diphosphate and those for the intermediate allylic diphosphate, which contains the cis-prenyl unit, are different during the cis-prenyl chain elongation reaction.     

Different kinetic patterns in the α-chymotrypsin-catalysed hydrolysis of synthetic ester substrates

The reaction of α-chymotrypsin with AcTyr-OEt and with AcTrp-OEt at pH 7.0 and 7.8 was studied over a wide range of substrate concentrations. The reaction with AcTyr-OEt at pH 7.8 was shown to be nonhyperbolic using a variety of criteria whereas those at pH 7.0 with the same substrate and at both pH values with AcTrp-OEt were hyperbolic. The non-hyperbolicity of the reaction with AcTyr-OEt at pH 7.8 followed a pattern of negative cooperativity with a Hill coefficient for the high substrate concentration range of 0.48. Although other explanations are possible, the pH dependence of the reaction with AcTyr-OEt could be related to the slow transition of the two known forms of the enzyme.Negative cooperativityNonhyperbolic kineticsα-ChymotrypsinHyperbolic kineticsHill coefficientSlow transition     

A new -2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

Half-site reactivity and the "induced-fit" hypothesis

Many subunit enzymes show the phenomenon of half-site reactivity, that is, the reaction with a substrate or substrate analogue shows a stoichiometry equal to one-half the number of “identical” subunits. One obvious potential explanation for this phenomenon is that reaction with one substrate molecule induces a change in an adjacent subunit, preventing a subsequent substrate molecule from reacting. Such an explanation has been proposed for the half-site reaction of the enzyme cytidine triphosphate synthetase with a substrate analogue (Levitzki et al., 1971). Analogous studies with glyceraldehyde-3-phosphate dehydrogenase reveal that the four active-site (Cys-149) sulfhydryl groups per tetrameric molecule react equivalently with iodoacetate whereas only two of the four sites undergo facile acylation with the substrate. The simple fact that the 2:1 stoichiometry ratio for the alkylation-acylation reactions is independent of the degree of prior alkylation rules out the ligand-induced asymmetry model as an explanation of the stoichiometries. Rather, it suggests that in muscle dehydrogenase there is a pre-existent non-equivalence among the subunits. On these bases we propose a procedure for distinguishing induced from pre-existent asymmetry in quaternary structure.     

Solvent and substrate deuterium isotope effects on a transamination reaction catalyzed by pig heart aspartate aminotransferase.

Transamination of erythro-β-hydroxy-l-aspartate catalyzed by pig heart aspartate aminotransferase (EC 2.6.1.1) was studied with both normal and α-deuterated substrate in $\text{H}{}_2\text{O}\text{D}{}_2\text{O}$. The overall transamination reaction, with α-ketoglutarate as amino group acceptor, showed no primary substrate isotope effect. However, one of the elementary reactions between two enzyme-substrate complexes was found to exhibit large primary isotope effects in both the forward and the reverse directions. This same reaction also showed a twofold solvent isotope effect in the reverse direction, but D₂O had only a negligible effect in the forward direction. These data were interpreted to indicate that the substrate α-hydrogen arises from a Bronsted acid with two equivalent hydrogens. Another elementary reaction, which is 100-fold slower, was also studied since it appeared to be one of the principal rate-determining steps in the overall reaction. This step was not affected by substrate deuteration but exhibited large solvent isotope effects in both directions.     

Kinetics of acetophenone reduction to (R)-1-phenylethanol by a whole-cell Pichia capsulata biocatalyst

(R)-1-phenylethanol is an important substance in fragrance and flavor industry. In this work, the reduction of acetophenone to (R)-1-phenylethanol in an aqueous medium was examined using Pichia capsulata as a whole-cell biocatalyst. Progress curve and initial rate measurements were used to obtain kinetic data. The experiments were carried out at pH 5, temperature of 25?°C, and in the presence of glucose to maintain in vivo regeneration of NADH. A model of the reversible reaction kinetics considering the substrate inhibition of the forward reaction was developed. Five kinetic parameters of this model were determined by a simultaneous fit of a reaction rate dependence on substrate concentration and 18 substrate and product concentration progress curves with very good accuracy. Equilibrium constant of the reaction and equilibrium conversion of acetophenone to (R)-1-phenylethanol were 13.7 and 93%, respectively.     

Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium

Racemic _DL-tert-leucine (_DL-Tle) was resolved to obtain enantiopure _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative with immobilized penicillin G acylase (PGA). The effects of pH, reaction temperature, substrate concentration and reaction time on the reaction were investigated. The reaction was conveniently carried out at 0.4 M substrate concentration in water at pH 8.0 and 30 °C. Under the optimized reaction conditions, _L-Tle was obtained in an enantiopure form (>99% ee) with 45.8% substrate conversion after 4 h. The thermal stability and operational stability of immobilized PGA were examined. Furthermore, the preparation of _L-Tle was successfully performed in a recirculating packed bed reactor (RPBR) system and immobilized PGA exhibited a long-term stability for 51 days with a slight decrease of activity. The isolated _D-enantiomer was racemized at 160 °C for 15 min and reused as substrate. The results obtained clearly demonstrated a potential for industrial application of immobilized PGA in the preparation of _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative.     

Investigation of factors affecting biotransformation of phytosterols to 9-hydroxyandrost-4-ene-3,-17-dione based on the HP-β-CD-resting cells reaction system

Abstract

Based on the hydroxypropyl-β-cyclodextrin-resting cells reaction system, high substrate concentration and improved reaction rate were achieved in a bioprocess of microbial side-chain cleavage of phytosterols to 9-OH-AD. Investigation of the kinetics of biotransformation showed that the initial reaction rate was strongly inhibited by a substrate concentration greater than 8 g/L. The oxygen transfer rate was also a critical parameter that could influence the rate of reaction. The results highlighted the importance of dissolved oxygen, particularly at high substrate concentration. Finally, analysis of metabolites showed that the main loss of process yield was due to by-product (16.3%) and nucleus degradation (8–11%) in the biotransformation. Investigation of the kinetics of metabolite formation provided fundamental knowledge on the biochemical reaction which was critical for understanding how to develop the biotransformation into an industrial application.     

Measurement of Allicin with N-Ethyl Maleimide

The reaction catalyzed by crystalline yeast phosphoglyceric acid mutase is inhibited by the substrate (d-2-phosphoglyceric acid). In order to elucidate the mechanism of this substrate inhibition, detailed investigations have been performed. It is proved that the substrate inhibition in this enzyme reaction is caused by the facts that the coenzyme-binding site on the enzyme is covered by the substrate and the combination of the coenzyme with the enzyme is interfered by the substrate. Consequently, it is concluded that the substrate is a competitive inhibitor of the coenzyme.     

The measurement process in biological systems: a new phenomenology.

A quantum theoretic approach to the problem of specific biological interactions at the molecular level, is presented. The concept of a “measuring system” in analogy with the enzyme macromolecule is used. The main hypothesis is that in the course of an enzymic reaction, the enzyme will specify the eigenvalues of the observables associated with the substrate, on some particular quantum states. Then, any “perturbation” induced in the substrate, will also be specified by the enzyme. In this context, the enzymic substrate is “perturbed” by an electromagnetic field and the physical transition S → S¹ thus induced is “measured” in the E_(S) + S¹ enzyme reaction, as compared with the control E_(S) + S reaction. The effect on the enzyme reaction is manifested by an enhancement of the reaction rate appearing periodically at well defined substrate irradiation times. The minimum substrate irradiation time inducing the first effect, termed t_m and the fixed time period that always appears to delimit two successive rate effects, termed the τ-parameter, are enzyme dependent. The same idea was used to devise an experimental model for the study of some more general interactions, within cellular systems. The growth of auxotrophic micro-organisms in minimal media supplemented with irradiated growth factors was followed. The pattern of growth stimulations obtained with this model, displays a similarity with the periodic enhancements of enzymic rates, obtained with irradiated substrates. This new type of evidence may suggest a characteristic of biological specificity, previously unrecognized.     

Biosynthesis of the widely distributed hydrocarbon (Z,Z)-6,9-heptadecadiene in astigmatid mites

ABSTRACT

Using a crude enzyme solution prepared from astigmatid mites, the conversion reaction to (Z,Z)-6,9-heptadecadiene (6,9-C17) using linoleyl aldehyde (LAld) as a substrate was successful. The mass spectrum of the reaction product using ¹³C-labeled LAld as a substrate could be assigned as ¹³C-labeled 6,9-C17. Unlike the findings in other species, the decarbonylase derived from mites did not require a coenzyme.     

Catenation and supercoiling in the products of bacteriophage λ integrative recombination in vitro

Catenanes (interlocked circular DNA molecules) are the exclusive products of the bacteriophage λ integrative recombination reaction in vitro when the substrate is a supercoiled DNA molecule containing both the attP and attB sites. It is proposed that the catenation results from the superhelical form of the substrate DNA. We also show that both circular DNA products of a single recombination event can be recovered as superhelical molecules with a superhelical density approximately that of the substrate DNA. The recombination reaction must therefore occur as a coupled process which does not permit free rotation around single-strand breaks at any stage.     

Kinetics of Thermal Inactivation of Lactate Dehydrogenase from Rabbit Muscle

The kinetics of thermal inactivation of rabbit muscle lactate dehydrogenase at different temperatures has been studied using the kinetic method for the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [Adv. Enzymol. Relat. Areas Mol. Biol. (1988), 61, 381–436]. The results show that thermal inactivation of the enzyme is an irreversible reaction. Microscopic rate constants were determined for thermal inactivation of the free enzyme and the enzyme–substrate complex. The inactivation rate constant of the free enzyme is much larger than the rate constant of the enzyme–substrate complex. The results suggest that the presence of the substrate has a certain protective effect against thermal inactivation of the enzyme.     

Alternative Substrate Kinetics of Escherichia coli Ribonuclease P: DETERMINATION OF RELATIVE RATE CONSTANTS BY INTERNAL COMPETITION*

A single enzyme, ribonuclease P (RNase P), processes the 5′ ends of tRNA precursors (ptRNA) in cells and organelles that carry out tRNA biosynthesis. This substrate population includes over 80 different competing ptRNAs in Escherichia coli. Although the reaction kinetics and molecular recognition of a few individual model substrates of bacterial RNase P have been well described, the competitive substrate kinetics of the enzyme are comparatively unexplored. To understand the factors that determine how different ptRNA substrates compete for processing by E. coli RNase P, we compared the steady state reaction kinetics of two ptRNAs that differ at sequences that are contacted by the enzyme. For both ptRNAs, substrate cleavage is fast relative to dissociation. As a consequence, V/K, the rate constant for the reaction at limiting substrate concentrations, reflects the substrate association step for both ptRNAs. Reactions containing two or more ptRNAs follow simple competitive alternative substrate kinetics in which the relative rates of processing are determined by ptRNA concentration and their V/K. The relative V/K values for eight different ptRNAs, which were selected to represent the range of structure variation at sites contacted by RNase P, were determined by internal competition in reactions in which all eight substrates were present simultaneously. The results reveal a relatively narrow range of V/K values, suggesting that rates of ptRNA processing by RNase P are tuned for uniform specificity and consequently optimal coupling to precursor biosynthesis.     

Osmoregulation inDunaliella. Catalysis of the glycerol-3-phosphate dehydrogenase reaction in a chloroplast-enriched fraction ofDunaliella tertiolecta

The glycerol-3-phosphate dehydrogenase (NAD-dependent) reaction was studied in a chloroplast-enriched fraction fromDunaliella tertiolecta. The reaction has widely separated pH optima for each direction. Reduction of dihydroxyacetone phosphate proceeded with Michaelis-Menten kinetics but sigmoidal double reciprocal plots were obtained with glycerol phosphate as variable substrate. NADP served as an alternative substrate but it was somewhat less effective than NAD. The reaction was inhibited by inorganic orthophosphate and by adenine nucleotides in a manner indicative of anion inhibition. Inhibition by inorganic phosphate was competitive with DHAP and possibly also with NADH. The enzyme was activated by Na⁺ at concentrations below 200 m and inhibited at higher concentrations, the region of maximum activation being affected by substrate concentration. Inhibition by Na⁺, present as a counterion of the substrate, was evidently responsible for apparent substrate inhibition by glycerol phosphate. Several important differences were apparent between the reaction in the unfractionated chloroplast-enriched fraction and the properties of a partly purified enzyme described by Haus and Wegmann (1984a, b).In toto, the results suggest that the regulatory potential of the reaction is probably more relevant to homeostatic control of glycerol content under steady state conditions than to controlling response to water stress.Abbreviations DHAP Dihydroxyacetone phosphate - CHES 2-(N-cyclohexylamino)ethanesulphonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Comprehensive Spectroscopic,Steady State,and Transient Kinetic Studies of a Representative Siderophore-associated Flavin Monooxygenase

Many siderophores used for the uptake and intracellular storage of essential iron contain hydroxamate chelating groups. Their biosyntheses are typically initiated by hydroxylation of the primary amine side chains of l-ornithine or l-lysine. This reaction is catalyzed by members of a widespread family of FAD-dependent monooxygenases. Here the kinetic mechanism for a representative family member has been extensively characterized by steady state and transient kinetic methods, using heterologously expressed N⁵-l-ornithine monooxygenase from the pathogenic fungus Aspergillus fumigatus. Spectroscopic data and kinetic analyses suggest a model in which a molecule of hydroxylatable substrate serves as an activator for the reaction of the reduced flavin and O₂. The rate acceleration is only ∼5-fold, a mild effect of substrate on formation of the C4a-hydroperoxide that does not influence the overall rate of turnover. The effect is also observed with the bacterial ornithine monooxygenase PvdA. The C4a-hydroperoxide is stabilized in the absence of hydroxylatable substrate by the presence of bound NADP⁺ (t_½ = 33 min, 25 °C, pH 8). NADP⁺ therefore is a likely regulator of O₂ and substrate reactivity in the siderophore-associated monooxygenases. Aside from the activating effect of the hydroxylatable substrate, the siderophore-associated monooxygenases share a kinetic mechanism with the hepatic microsomal flavin monooxygenases and bacterial Baeyer-Villiger monooxygenases, with which they share only moderate sequence homology and from which they are distinguished by their acute substrate specificity. The remarkable specificity of the N⁵-l-ornithine monooxygenase-catalyzed reaction suggests added means of reaction control beyond those documented in related well characterized flavoenzymes.     

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile via asymmetric transcyanation of acetyltrimethylsilane with acetone cyanohydrin in an aqueous/organic biphasic system catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal was successfully carried out for the first time. The optimal volume ratio of aqueous to organic phase, buffer pH value and reaction temperature were 15% (v/v), 5.0 and 30°C, respectively, under which both substrate conversion and product enantiomeric excess (ee) were 99%. Silicon atom in the substrate showed great effect on the reaction. Acetyltrimethylsilane was a much better substrate for (R)-hydroxynitrile lyase from Prunus japonica than its carbon analogue.     

β-galactosidase-catalyzed synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol: Optimization by response surface methodology

In this study, the synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol (GG) was performed by the reverse hydrolysis of D-galactose and glycerol using β-galactosidase from Kluyveromyces lactis. Four process variables, reaction temperature (30.0–45.0?°C), reaction time (24–48?h), enzyme concentration (150.00–350.00?U/mL), and substrate molar ratio (glycerol:D-galactose, 7.5:12.5?mmol/mmol) were investigated and optimized via response surface methodology (RSM) for optimal GG synthesis. Both quadratic equations and the optimal reaction conditions were established. Results showed that the four variables, i.e., reaction temperature, reaction time, enzyme concentration, and substrate molar ratio had significant (p?β-galactosidase concentration and 8.65:1.00 of substrate molar concentration ratio (glycerol: D-galactose) at 39.8?°C and 48?h of reaction. Under these conditions, the GG concentration was 140.03?g/L and GG yield was 55.71%, which both were close to the predicted values (143.26?g/L and 56.73%). This finding proves the RSM to be a useful tool in optimizing process conditions for GG synthesis.     

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

The proteomic mapping of enzyme–substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide‐based substrate analog capable of reprogramming the wild‐type phosphoryl‐transfer reaction to produce a kinase‐acrylamide‐based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP‐based methacrylate crosslinker (ATP‐MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP‐MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP‐based methacrylate (ADP‐MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP‐MA over ATP‐MA is due to reduced inhibition of the second step of the kinase–substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP‐MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site‐specific manner. The kinase–substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.